Approaches for the Development of Serum-Free Media

Top-Down Approach for Serum Replacement

There are two basic approaches to developing serum-free media formulations. The first approach (top-down) might involve selecting an existing formulation for a similar cell line, supplemented with serum to obtain a reasonable level of growth. The content of serum could then be gradually reduced to cause a decrease in the cell growth to say 50 % of that originally obtained. At this point selected components could be added to the medium to restore the original level of cell growth.

The top-down approach is often easier to pursue since a working serum-free formulation can often be developed more quickly. Cell lines that belong to the same group, such as epithelial or transformed, often require the same growth factors for growth. Therefore a formulation that works for one epithelial cell line may work for another with minimal modifications to certain growth factors or hormones. For this reason serum-free formulations can be designed faster by this approach.

This method involves a systematic approach for the gradual replacement of serum by substitution with essential nutrients or growth factors. In this procedure the concentration of serum in gradually reduced to a level which around 50 % of maximal growth as measured by the cell yields after 3 or 4 days. This procedure may involve the adaptation of cells to gradually reduced levels of serum. Typically adaptation can be successful in reducing the serum concentration from 10 % to 2 %. At the lower serum level the growth rate will decrease until appropriate supplements are provided in the medium or until the cells adapt. Cellular adaptation can involve the synthesis of essential components by the cells.

The drawback to the top-down approach is that many components in the formulation may be unnecessary, and often inhibitory for growth. This can often result in the “capping” of the optimal performance of the medium (i.e.: the maximum growth may not be achieved) as improvements are hindered by the presence of unwanted compounds.

Bottom-Up Approach for Serum Replacement

The bottom-up approach involves the addition of potentially growth promoting components systematically to the media in an attempt to obtain incremental increases in cell growth. Although more labour-intensive and time-consuming, this approach can lead to higher quality media. Only the components that are required for growth are included in the formulation, allowing for greater control of optimizing the medium. Thus media developed in this way tend to have higher growth rates and are more easily improved since inhibitory compounds are less likely to be present.

Fig. 8.2 The determination of the optimal concentration of a component

There is likely to be a wide concentration range over which any nutrient supplement is not limiting. Using Ham's approach (Ham and McKeehan 1979) the optimum concentration for the nutrient can be set at the mid-point of the broad optimum plateau of the growth-response curve (Fig. 8.2). This enables the concentration to be bracketed between a maximum and minimum value. This reduces the likelihood that the component will become growth limiting through the adjustments of other medium components. In the response curve shown in Fig. 8.2 the mid-point of the optimum concentration is 10 5 M. The concentration range of a supplement can be established initially by testing a broad range at tenfold increases e.g.: x0.1, x1, x10 and x100 of a nominal starting concentration. This can be followed by focus on a narrower range to obtain the optimum concentration for growth.

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